Impairment of Nitric Oxide Pathway by Intravascular Hemolysis Plays A
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1521-0103/367/2/194–202$35.00 https://doi.org/10.1124/jpet.118.249581 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 367:194–202, November 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Impairment of Nitric Oxide Pathway by Intravascular Hemolysis Plays a Major Role in Mice Esophageal Hypercontractility: Reversion by Soluble Guanylyl Cyclase Stimulator Fabio Henrique Silva, Kleber Yotsumoto Fertrin, Eduardo Costa Alexandre, Fabiano Beraldi Calmasini, Carla Fernanda Franco-Penteado, and Fernando Ferreira Costa Hematology and Hemotherapy Center (F.H.S., K.Y.F., C.F.F.-P., F.F.C.) and Department of Pharmacology, Faculty of Medical Sciences (E.C.A., F.B.C.), University of Campinas, Campinas, São Paulo, Brazil; and Division of Hematology, University of Washington, Seattle, Washington (K.Y.F.) Downloaded from Received April 1, 2018; accepted July 30, 2018 ABSTRACT Paroxysmal nocturnal hemoglobinuria (PNH) patients display cyclase stimulator 3-(4-amino-5-cyclopropylpyrimidin-2-yl)- exaggerated intravascular hemolysis and esophageal disor- 1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY 41-2272; ders. Since excess hemoglobin in the plasma causes re- 1 mM) completely reversed the increased contractile responses jpet.aspetjournals.org duced nitric oxide (NO) bioavailability and oxidative stress, we to CCh, KCl, and EFS in PHZ mice, but responses remained hypothesized that esophageal contraction may be impaired unchanged with prior treatment with NO donor sodium nitro- by intravascular hemolysis. This study aimed to analyze the prusside (300 mM). Protein expression of 3-nitrotyrosine and alterations of the esophagus contractile mechanisms in a 4-hydroxynonenal increased in esophagi from PHZ mice, sug- murine model of exaggerated intravascular hemolysis induced gesting a state of oxidative stress. In endothelial nitric oxide by phenylhydrazine (PHZ). For comparative purposes, sickle synthase gene-deficient mice, the contractile responses elicited cell disease (SCD) mice were also studied, a less severe by KCl and CCh were increased in the esophagus but remained intravascular hemolysis model. Esophagus rings were dis- unchanged with the intravascular hemolysis induced by PHZ. In at ASPET Journals on September 30, 2021 sected free and placed in organ baths. Plasma hemoglobin conclusion, our results show that esophagus hypercontractile was higher in PHZ compared with SCD mice, as expected. The state occurs in association with lower NO bioavailability due to contractile responses produced by carbachol (CCh), KCl, and exaggerated hemolysis intravascular and oxidative stress. electrical-field stimulation (EFS) were superior in PHZ esophagi Moreover, our study supports the hypothesis that esophageal compared with control but remained unchanged in SCD mice. disorders in PNH patients are secondary to intravascular hemo- Preincubation with the NO-independent soluble guanylate lysis affecting the NO-cGMP pathway. Introduction The gastrointestinal tract consists of a muscular tube that extends from the mouth to the anus. The esophagus is part of – The nitric oxide (NO) cGMP signaling pathway is well the gastrointestinal tract, and its function is transporting known to regulate smooth muscle relaxation and contraction swallowed food into the stomach and preventing gastroesoph- (Förstermann and Sessa, 2012). Upon stimulation by NO, ageal reflux (Yazaki and Sifrim, 2012). Esophagus smooth the smooth muscle soluble guanylate cyclase (sGC) enzyme muscle receives a rich cholinergic excitatory innervation, catalyzes the production of cGMP following activation of resulting in the discharge of acetylcholine that acts on the cGMP-dependent protein kinase, which leads to smooth muscarinic receptor of smooth muscle and promoting esoph- muscle relaxation from different kinds of tissues (Francis ageal smooth muscle contractions (Goyal and Chaudhury, et al., 2010; Förstermann and Sessa, 2012). Alterations of 2008; Braverman et al., 2009). NO produced by nitrergic the NO signaling pathway in the vascular or nonvascular nerves is considered the principal inhibitory neurotransmitter smooth muscle have been associated with several disorders, that promotes smooth muscle relaxation of the esophagus such as cardiovascular disease and gastrointestinal disease (Shah et al., 2004; Goyal and Chaudhury, 2008). A previous (Mearin et al., 1993; Förstermann and Sessa, 2012). study showed mRNA expression of endothelial nitric oxide synthase in human esophageal mucosa (Inamori et al., 2006). Esophagus contraction induced by cholinergic nerve stimula- F.H.S. and F.F.C. thank São Paulo Research Foundation (FAPESP) for financial support [Grants 2014/00984-3, 2017/08122-9, and 2018/06243-6]. tion is increased by NO production inhibition, as well as https://doi.org/10.1124/jpet.118.249581. esophageal peristalsis (Yamato et al., 1992; Lecea et al., 2011). ABBREVIATIONS: BAY 41-2272, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine; CCh, carbachol; EFS, electrical-field stimulation; eNOS2/2, endothelial nitric oxide synthase gene-deficient; 4-HNE, 4-hydroxynonenal; NO, nitric oxide; 3-NT, 2 3-nitrotyrosine; O2 , superoxide anion; pEC50, potency; PHZ, phenylhydrazine; PNH, paroxysmal nocturnal hemoglobinuria; SCD, sickle cell disease; sGC, soluble guanylate cyclase; SNP, sodium nitroprusside. 194 Effects of Intravascular Hemolysis on Esophagus 195 A previous study reported that guanylyl cyclase gene–deficient We injected PHZ at 50 mg/kg into C57BL/6 and eNOS2/2 mice mice display higher esophagus contractions compared with wild intraperitoneally to induce intravascular hemolysis. Mice were rein- mice (Groneberg et al., 2015). jected with 50 mg/kg 8 hours later (Lim et al., 1998) and were killed Paroxysmal nocturnal hemoglobinuria (PNH) is a rare with inhaled isoflurane 4 days after the end of PHZ treatment. acquired clonal hematopoietic stem cell disease in which C57BL/6 and PHZ mice received deferoxamine (150 mg/kg per day via i.p. injection, from second to fourth day), deferiprone (50 mg/kg per uncontrolled complement activation leads to intravascular day, given as daily gavage from second to fourth day), or vehicles hemolysis (Oni et al., 1970; Hill et al., 2017). PNH patients (saline and water, respectively). display dysphagia/odynophagia, and these alterations seem to Hematologic Parameters. Whole blood was obtained by intra- be due to esophageal spasm, but the mechanisms underlying cardiac puncture from ketamine/xylazine-anesthetized mice and anti- esophageal dysfunction are largely unknown (Moyo et al., coagulated with ethylenediamine tetraacetic acid for blood count 2004; Parker et al., 2005). Sickle cell disease (SCD) is also a determination within 30–60 minutes of blood collection on Sysmex blood disorder that manifests with hemolytic anemia, but to XN-3000 equipment (Sysmex, Kobe, Japan). the best of our knowledge, SCD patients do not display Lactate Dehydrogenase and Plasma Hemoglobin Measure- esophageal disease. In PNH and SCD, intravascular hemoly- ment. Mice plasma samples were used to quantify the lactate dehy- sis is a common hallmark, but this clinical evidence is less drogenase and hemoglobin through colorimetric assays using the severe in SCD than in PNH (Reiter et al., 2002; Hill et al., Hemoglobin Colorimetric Assay (Cayman Chemical, Ann Arbor, Michigan) and Bilirubin Assay Kit (Sigma-Aldrich, St. Louis, MO) 2010). In intravascular hemolysis, erythrocyte lysis in the ’ according to the manufacturer s procedure. Assays were done in Downloaded from circulation leads to release of free hemoglobin and other duplicate. erythrocyte components into the plasma, such as asymmetric Functional Studies in Esophagus Smooth Muscle Rings and dimethylarginine, arginase-1, and lactate dehydrogenase Concentration-Response Curves. The esophagus was extracted, (Reiter et al., 2002; Hill et al., 2010; Kato et al., 2017). It is dissected, and cut into rings (1.5–2 mm in length). Rings of esophagus well established that NO scavenging by an excess of plasma smooth muscle were placed in a 5-ml organ system filled with Krebs hemoglobin contributes to several clinical complications of solution at 37°C continuously bubbled with a mixture of 95% O2 and intravascular hemolysis (Hill et al., 2010; Kato et al., 2017). 5% CO2 (pH 7.4). Isometric force was analyzed using a Power jpet.aspetjournals.org The contribution of oxidative stress to the deleterious effects Laboratory version 7.2 system (ADInstruments, Sydney, Australia). of intravascular hemolysis has been previously shown (Fibach The esophagus was allowed to equilibrate for 60 minutes under a resting tension of 1 mN. and Dana, 2015). A result of oxidative stress is the inactivation 2 Cumulative concentration-response curves for the muscarinic of NO biologic activity by superoxide anion (O2 ) excess agonist carbachol (CCh; 10 nM to 100 mM) and KCl (1 mM to 1 M) (Pacher et al., 2007). In this study, since cell-free hemoglobin were acquired in esophagus rings from control, PHZ-treated C57BL/6, excess and oxidative stress reduce the amount of bioavailable SCD, eNOS2/2, and PHZ-treated eNOS2/2 mice. Esophagus con- NO, we hypothesized that exaggerated intravascular he- tractile responses to these compounds were acquired in the absence at ASPET Journals on September 30, 2021 molysis could damage normal smooth muscle function in the and presence of NO-independent sGC stimulator 3-(4-amino-5- esophagus. We aimed to evaluate the